CN111840650B - Collagen-based modified citrus pectin composite material and preparation method and application thereof - Google Patents
Collagen-based modified citrus pectin composite material and preparation method and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
Abstract
The invention relates to a collagen-based modified citrus pectin composite material and a preparation method and application thereof. The collagen-based modified citrus pectin composite material is prepared by taking collagen and modified citrus pectin as raw materials, firstly preparing a collagen membrane material and preparing a modified citrus pectin solution, further loading the modified citrus pectin on the collagen membrane material by adopting an adsorption method, and adjusting the concentration and the pH value of the modified citrus pectin solution to enable the adsorption amount of the collagen material per square centimeter to the modified citrus pectin to be adjustable within the range of 0.01-10mg so as to obtain the collagen-based modified citrus pectin composite material with different loading amounts. The material can be in a porous three-dimensional structure with different loosening degrees, has good biocompatibility, and can be applied to the regeneration and repair of guide induction tissues.
Description
Technical Field
The invention belongs to the field of biomedical composite materials, and particularly relates to a collagen-based modified citrus pectin composite material as well as a preparation method and application thereof.
Background
Tissue disease defects caused by wounds, infection, tumors, degenerative changes and the like are main hazards affecting human health, and the tissue regeneration and repair material provides an effective way for tissue repair and reconstruction. Meanwhile, the tissue injury repair process is inevitably accompanied by the occurrence of inflammatory reaction, which is accompanied by the whole process of tissue regeneration and repair, scarring, and the like. Therefore, there is a need for the development of a tissue regeneration repair material having a function of regulating immune inflammation of damaged tissues.
Collagen is a main component constituting extracellular matrix, has a natural triple helix structure, is a natural degradable biomaterial with good cell and biocompatibility, low immunogenicity, low toxicity, good biochemical and physical mechanical properties, can promote cell growth and adhesion, and has been widely applied to the regeneration and repair of various tissues such as bone, cartilage, skin, nerve, tendon, ligament and the like. Meanwhile, the collagen can also be used as various bioactive substances, growth factors and drug carriers to protect the activity of bioactive molecules and realize the controlled and sustained release of the bioactive molecules. The constructed collagen-based composite material compounded with various growth factors has been widely researched and applied in tissue injury repair. The novel collagen-based composite material is constructed by loading active molecules with immunoregulation function, and a new way can be provided for developing tissue regeneration repair materials with the function of regulating and controlling the immune inflammation of damaged tissues.
Pectin also has clear application prospects in tissue repair. Chinese patent CN111218421A discloses an adhesion type cell scaffold and preparation and application thereof, which relate to a preparation method and application of a cell adhesion type scaffold taking graft modified aminated citrus pectin as a base material, and show application values in aspects of pectin reorganization and regeneration repair and tissue engineering. It is also noteworthy that Modified Citrus Pectin (MCP) obtained by chemical, biological, physical methods with a molecular weight below 35 kilodaltons (kDa) and a degree of esterification below 30% can exert immunomodulatory effects by activating T helper/inducer cells, B cells and natural phagocytic (NK) cells. In particular, MCP is a natural antagonist of the proinflammatory cytokines galactoside lectin-3 (Galectin-3, Gal-3). Gal-3 has important immune inflammation regulation and control functions in the processes of tissue injury repair, chronic inflammation, tissue fibrosis and the like; moreover, it is also involved in the regulation of various cancer cells, promoting tumor formation, development and metastasis. Therefore, the regulation of the immune inflammatory response of tissues by inhibiting the biological activity of Gal-3 has become a new strategy for the treatment of various diseases. MCP not only can competitively inhibit the action of Gal-3 by combining with cell surface receptors of Gal-3, but also directly inhibits the expression of Gal-3, and has the characteristics of anticancer, heavy metal adsorption, anti-inflammatory, immunoregulation, blood fat reduction, drug transport and the like, excellent biodegradability, biocompatibility, safety, no toxicity, low cost and the like.
At present, the modified citrus pectin is mainly used by an oral aqueous solution way, the effective local action concentration is difficult to achieve in the process of repairing the defected tissues, and an effective way can be provided for solving the problem by preparing the composite material which can be implanted and loaded with the modified citrus pectin, so that the modified citrus pectin in the damaged area achieves the effective action concentration. Because the collagen and the modified citrus pectin respectively have a large number of amino groups, carboxyl groups, hydroxyl groups and other groups, the collagen-based composite material loaded with the modified citrus pectin can be prepared by utilizing the interaction of hydrogen bonds, static electricity and the like between the collagen and the modified citrus pectin through a simple adsorption means, and a tissue-guided induced regeneration repair material with local immune regulation, anti-inflammation and anti-tissue fibrosis functions is formed.
Disclosure of Invention
The invention aims to provide a collagen-based modified citrus pectin composite material and a preparation method and application thereof, so that the characteristics of the modified citrus pectin and the collagen material are combined to form a tissue-guided regeneration-inducing repair material with local immunoregulation, anti-inflammation and anti-tissue fibrosis functions. The material directly acts on the injured part to play the effective role of the modified citrus pectin and form the tissue-guided regeneration-inducing repair material with the functions of local immune regulation, anti-inflammation and anti-tissue fibrosis.
The collagen-based modified citrus pectin composite material provided by the invention is prepared by taking collagen and modified citrus pectin as raw materials, the modified citrus pectin is loaded on a collagen membrane material by adopting an adsorption method, and the adsorption amount of the collagen material per square centimeter to the modified citrus pectin is adjustable within the range of 0.01-10 mg.
In the collagen-based modified citrus pectin composite material, the loading amount of the modified citrus pectin can be regulated and controlled by adjusting the concentration and pH value of the modified citrus pectin.
In the collagen-based modified citrus pectin composite material, the thickness of the collagen film is 0.5-3 mm.
The preparation method of the collagen-based modified citrus pectin composite material provided by the invention comprises the following steps:
1) dissolving modified citrus pectin powder in phosphate buffer solution or Tris alkali buffer solution, and magnetically stirring for 0.5-1h to obtain MCP solution of 0.01-10 mg/ml;
2) dissolving collagen in 0.3% malonic acid solution, swelling at 4 deg.C for 1 week, and stirring to obtain 0.1-1.0% collagen swelling solution;
3) pre-freezing the collagen swelling solution at-80 deg.C to-40 deg.C, and freeze-drying, or air-drying and flattening to obtain collagen films with compact structure and different loosening degrees;
4) and (2) placing the prepared collagen membrane material into the modified citrus pectin solution, reacting for 5-6 hours at 25-30 ℃, pre-freezing the collagen-based material at-80 ℃ -40 ℃, then carrying out secondary freeze drying, washing with deionized water to remove the non-adsorption-reacted modified citrus pectin and solvent molecules, and carrying out freeze drying again to obtain the final collagen-based modified citrus pectin composite material.
The modified citrus pectin has a molecular weight of less than 35kDa and an esterification degree of less than 30 percent.
The collagen swelling solution is collagen type I derived from tendon of cattle, or collagen of other animal such as rat tail.
Preferably, the collagen swelling solution has a mass volume concentration of 0.6%.
The pH value of the modified citrus pectin solution is 5-10.
The modified citrus pectin solution is ready for use.
The collagen-based modified citrus pectin composite material provided by the invention can be used for a tissue injury repair material, so that the collagen membrane loaded modified citrus pectin directly acts on an injured part to play an effective role of the modified citrus pectin, and tissue-guided induced regeneration with local immune regulation, anti-inflammation and anti-tissue fibrosis functions is formed.
The invention provides a collagen-based modified citrus pectin composite material and a preparation method and application thereof. Collagen with good biocompatibility and modified citrus pectin serving as a Gal-3 inhibitor are used as raw materials to prepare the tissue damage repair material, the collagen membrane loaded modified citrus pectin directly acts on a damaged part to play the effective role of the modified citrus pectin, and the tissue-guided induced regeneration repair material with local immune regulation, anti-inflammation and anti-tissue fibrosis functions is formed.
Drawings
FIG. 1a is a scanning electron micrograph of a composite obtained in accordance with the first example;
FIG. 1b is a scanning electron micrograph of the composite obtained in example III;
FIG. 2 is a graph of a standard curve of modified citrus pectin obtained by the method used in example two;
FIG. 3 is a graph of MCP adsorption amount of a collagen-based modified citrus pectin composite material from example one 10mg/ml MCP (in Tris base buffer pH = 10);
fig. 4 is a graph of MCP adsorption of collagen-based modified citrus pectin composites from example three 5mg/ml MCPs (in PBS buffer pH = 7).
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example one
(1) The bovine tendon collagen is extracted according to the method of the inventor's prior Chinese patent CN 107412869B, and is stored after freeze drying. Weighing 6g of collagen, dissolving the collagen in 1000ml of malonic acid with the volume concentration of 0.3%, swelling for 1 week at the temperature of 4 ℃, uniformly stirring by using a tissue homogenizer, centrifuging at 1200r/min for 20 minutes to remove bubbles, and finally obtaining the collagen swelling solution with the mass concentration of 0.6%.
(2) Modified citrus pectin powder (1 g, Zhejiang fruit source health products Biotech Co., Ltd.) was weighed and dissolved in 100ml of 0.1M Tris buffer (pH = 10), and the solution was magnetically stirred for 1 hour to obtain a solution of 10 mg/ml.
(3) Putting a certain amount of collagen swelling solution into a mold, freezing at-40 ℃, and freeze-drying to obtain a formed collagen film with the thickness of about 2 mm; cutting collagen membrane into 1 × 1cm2Adding 1.5ml of MCP solution, reacting at 30 ℃ for 6h, freeze-drying (at-40 ℃), washing with deionized water, and freeze-drying to obtain the final collagen-based MCP composite material.
(4) And (3) measuring the quality of the citrus pectin modified by collagen film adsorption by using a m-hydroxybiphenyl method. The adsorption quantity of the collagen-based material MCP is 7.954-8.834 mg/cm2。
Example two
The specific method for measuring the quality of the citrus pectin modified by collagen film adsorption by using the m-hydroxybiphenyl method comprises the following steps:
(1) collecting all cleaning liquid in the process of adsorbing the MCP solution by the collagen membrane, freeze-drying, adding 5ml of deionized water for dissolving, measuring the ultraviolet absorption at 525nm by using a m-hydroxybiphenyl method, and calculating the concentration of the non-adsorbed MCP by taking a standard curve of the MCP solution as a comparison.
(2) 0.15 g of m-hydroxybiphenyl is weighed and dissolved in 5mg/mL of sodium hydroxide solution, the volume is determined to be 100mL, and the mass concentration of the m-hydroxybiphenyl solution is 1.5 mg/mL.
(3) 0.478 g of sodium tetraborate is weighed and dissolved in 100mL of concentrated sulfuric acid to obtain a sodium tetraborate/sulfuric acid solution for later use.
(4) Preparation of MCP standard curve: accurately measuring MCP solution (1 mg/mL) 0.1, 0.3, 0.5, 0.7, 0.9 and 1mL, placing the MCP solution in a 10 mL volumetric flask, adding water to a constant volume, accurately measuring each solution 0.5mL to 10 mL test tubes with stoppers respectively, placing the test tubes in an ice water bath, adding 2.5 mL sodium tetraborate/sulfuric acid solution, uniformly mixing the solution with a vortex mixer, heating the solution in a boiling water bath for 5 min after all the solution is added, adding 1.5mg/mL m-hydroxybiphenyl solution 50ul by using a trace sample adding gun after cooling the solution in the ice water bath, shaking the solution for 5 min after uniform mixing, and removing bubbles by ultrasound. The above procedure was followed with 0.5mL of water to obtain a blank solution, and the UV absorbance was measured at a wavelength of 525nm to obtain the regression equation of the standard curve.
(5) And (3) measuring the ultraviolet absorption of the unadsorbed liquid obtained in the step (1) at the wavelength of 525nm according to the operation of the step (4), and calculating the concentration of the unadsorbed MCP by taking a standard curve of the MCP solution as a comparison.
EXAMPLE III
(1) Weighing 6g of collagen, dissolving the collagen in 1000ml of malonic acid with the volume concentration of 0.3%, swelling for 1 week at 4 ℃, uniformly stirring by using a tissue homogenizer, centrifuging at 1200r/min for 20 minutes to remove bubbles, and finally obtaining the collagen swelling solution with the mass concentration of 0.6%.
(2) MCP (modified citrus pectin) powder was weighed out at 500mg, dissolved in 100ml of 0.01M PBS buffer (pH = 7), and magnetically stirred for 1h to obtain MCP solution at 5 mg/ml.
(3) Putting a certain amount of collagen swelling solution into a mold, freezing at-40 ℃, and freeze-drying to obtain a formed collagen film with the thickness of about 2 mm; cutting collagen membrane into 1 × 1cm2Adding 1.5ml of MCP solution, reacting at 30 ℃ for 6h, freeze-drying, washing with deionized water, and freeze-drying to obtain the final collagen-based MCP composite material.
(4) And (3) measuring the quality of the citrus pectin modified by collagen film adsorption by using a m-hydroxybiphenyl method. The adsorption quantity of the MCP of the collagen-based material is 3.218-4.574 mg/cm2。
FIG. 1a is the SEM image of the composite material obtained in the first example, and FIG. 1b is the SEM image of the composite material obtained in the third example. As can be seen from the figure, the composite material has a loose and porous three-dimensional structure, and the pore diameters are communicated with each other.
FIG. 2 is a standard curve of modified citrus pectin obtained by the method used in example two.
Fig. 3 is a graph of MCP adsorption of the collagen-based modified citrus pectin composite material obtained in example one.
Fig. 4 is a graph of MCP adsorption of the collagen-based modified citrus pectin composite obtained in example three.
The collagen-based modified citrus pectin composite material has good pore structure and porosity, the modified citrus pectin is loaded on a collagen membrane to achieve the effective concentration, and the loading amount of the modified citrus pectin can be controlled.
In light of the above-described embodiments of the present invention, various changes and modifications can be made by the worker who works the present invention without departing from the scope of the present invention. The technical scope of the present invention is not limited to the contents of the specification, and must be determined according to the scope of the claims.
Claims (9)
1. A collagen-based modified citrus pectin composite material is characterized in that the collagen-based modified citrus pectin composite material is prepared by taking collagen and modified citrus pectin as raw materials, the modified citrus pectin is loaded on a collagen membrane material by adopting an adsorption method, and the adsorption amount of the collagen membrane material per square centimeter to the modified citrus pectin is adjustable within the range of 0.01-10 mg; the preparation method comprises the following steps:
1) dissolving modified citrus pectin powder in phosphate buffer solution or Tris alkali buffer solution, and magnetically stirring for 0.5-1h to obtain MCP solution of 0.01-10 mg/mL;
2) dissolving collagen sponge in 0.3% malonic acid solution, swelling at 4 deg.C for 1 week, and stirring to obtain 0.1-1.0% collagen swelling solution;
3) pre-freezing the collagen swelling solution at-80 deg.C to-40 deg.C, and freeze-drying, or air-drying and flattening to obtain collagen films with compact structure and different loosening degrees;
4) and (2) placing the prepared collagen membrane material into the modified citrus pectin solution, reacting for 5-6 hours at 25-30 ℃, pre-freezing the collagen-based material at-80 ℃ -40 ℃, then carrying out secondary freeze drying, washing with deionized water to remove the non-adsorption-reacted modified citrus pectin and solvent molecules, and carrying out freeze drying again to obtain the collagen-based modified citrus pectin composite material.
2. The collagen-based modified citrus pectin composite according to claim 1, wherein the collagen-based modified citrus pectin composite has a collagen film thickness of 0.5 to 3 mm.
3. A method of preparing a collagen-based modified citrus pectin composite according to claim 1, comprising the steps of:
1) dissolving modified citrus pectin powder in phosphate buffer solution or Tris alkali buffer solution, and magnetically stirring for 0.5-1h to obtain MCP solution of 0.01-10 mg/mL;
2) dissolving collagen sponge in 0.3% malonic acid solution, swelling at 4 deg.C for 1 week, and stirring to obtain 0.1-1.0% collagen swelling solution;
3) pre-freezing the collagen swelling solution at-80 deg.C to-40 deg.C, and freeze-drying, or air-drying and flattening to obtain collagen films with compact structure and different loosening degrees;
4) and (2) placing the prepared collagen membrane material into the modified citrus pectin solution, reacting for 5-6 hours at 25-30 ℃, pre-freezing the collagen-based material at-80 ℃ -40 ℃, then carrying out secondary freeze drying, washing with deionized water to remove the non-adsorption-reacted modified citrus pectin and solvent molecules, and carrying out freeze drying again to obtain the final collagen-based modified citrus pectin composite material.
4. The method of claim 3, wherein the modified citrus pectin has a molecular weight of less than 35kDa and a degree of esterification of less than 30%.
5. The method according to claim 3, wherein the swelling solution of collagen is collagen type I derived from bovine tendon, or collagen derived from other animal species.
6. The method according to claim 3, wherein the collagen swelling solution of step 2) has a mass/volume concentration of 0.6%.
7. The method of claim 3, wherein the modified citrus pectin solution of step 1) has a pH of from 5 to 10.
8. The method according to claim 3, wherein the lyophilization temperature in the steps 3) and 4) is-40 ℃.
9. The method of claim 3, wherein the modified citrus pectin solution is ready for use.
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